Systems, methods and assays for outlier clustering unsupervised learning automated report (OCULAR)

This application is the U.S. national phase of PCT Appln. No. PCT/US2020/055575 filed Oct. 14, 2020, which claims the benefit of U.S. provisional application Ser. No. 62/914,763 filed Oct. 14, 2019, the disclosure of which is hereby incorporated in its disclosures of which are hereby incorporated in their entirety by reference herein.

This invention was made with government support under. CA143906 awarded by the National Institutes of Health. The government has certain rights in the invention.”

In at least one aspect, a system for identification of biological structures present in a liquid biopsy sample based on fluorescence characteristics and morphology of biological structures is provided. In another aspect, a method and an assay for identification of the biological structures present in a liquid biopsy sample is provided. Additional aspects further relate to diagnosis and treatment of a human afflicted with a disease by using the systems, methods and assays set forth herein.

Traditionally, the field of rare event detection in cancer (often referred to as liquid biopsy or circulating tumor cells) has focused on certain rare cell subtypes with known or presumed distinct phenotypes through various enrichment methods that select a specific target cell based on its presumably known phenotype (e.g. a protein based phenotype; a shape based phenotype; or other biophysical, physical, chemical based phenotype).

The cancer and its related cells, however, appear to be much more complex and varied than traditionally recognized, and the rare cells in the blood may not neatly fall into a specific category and/or consistent categories.

The following publications are related art for the background of this disclosure: Allard, W. J., et al., Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res, 2004. 10(20): p. 6897-904; Liu, C.-Y., et al., Vimentin contributes to epithelial-mesenchymal transition cancer cell mechanics by mediating cytoskeletal organization and focal adhesion maturation. Oncotarget, 2015. 6(18); Vuoriluoto, K., et al., Vimentin regulates EMT induction by Slug and oncogenic H-Ras and migration by governing Axl expression in breast cancer. Oncogene, 2011. 30(12): p. 1436-1448; Lou, X.-L., et al., Interaction between circulating cancer cells and platelets: clinical implication. Chinese journal of cancer research=Chung-kuo yen cheng yen chiu, 2015. 27(5): p. 450-460; Goubran, H. A., et al., Platelets effects on tumor growth. Semin Oncol, 2014. 41(3): p. 359-69; Li, N., Platelets in cancer metastasis: To help the “villain” to do evil. International Journal of Cancer, 2016. 138(9): p. 2078-2087; Labelle, M., S. Begum, and Richard O. Hynes, Direct Signaling between Platelets and Cancer Cells Induces an Epithelial-Mesenchymal-Like Transition and Promotes Metastasis. Cancer Cell, 2011. 20(5): p. 576-590; Boraas, L. C. and T. Ahsan, Lack of vimentin impairs endothelial differentiation of embryonic stem cells. Scientific Reports, 2016. 6: p. 30814; Miettinen, M. and J. F. Fetsch, Distribution of keratins in normal endothelial cells and a spectrum of vascular tumors: Implications in tumor diagnosis. Human Pathology, 2000. 31(9): p. 1062-1067; Dellagi, K., et al., Expression of vimentin intermediate filament cytoskeleton in acute nonlymphoblastic leukemias. Blood, 1985. 65(6): p. 1444-52; Gustmann, C., et al., Cytokeratin expression and vimentin content in large cell anaplastic lymphomas and other non-Hodgkin's lymphomas. Am J Pathol, 1991. 138(6): p. 1413-22; Satelli, A., et al., Epithelial-mesenchymal transitioned circulating tumor cells capture for detecting tumor progression. Clinical cancer research: an official journal of the American Association for Cancer Research, 2015. 21(4): p. 899-906; Yang, S. and X. Li, Recent advances in extracellular vesicles enriched with non-coding RNAs related to cancers. Genes & diseases, 2017. 5(1): p. 36-42; and Thery, C., et al., Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of extracellular vesicles, 2018. 7(1): p. 1535750-1535750; Zeuschner, P., J. Linxweiler, and K. Junker, Non-coding RNAs as biomarkers in liquid biopsies with a special emphasis on extracellular vesicles in urological malignancies. Expert Review of Molecular Diagnostics, 2019: p. 1-17.

Accordingly, there is a need for improved techniques in detecting and characterizing rare and common events in a liquid biopsy sample.

In at least one aspect, a system for identification of a biological structure present in a liquid biopsy sample is provided. The system identifies common biological structures and rare biological structures based on their fluorescence characteristics and morphology. The identified biological structures can be used in the diagnosis and treatment of a human afflicted with a disease. Examples described in this disclosure also relate to methods and assays that may be used together with the systems of this disclosure for diagnosis and treatment of a human afflicted with a disease.

In another aspect, a system for identification of a biological structure (“biological structure identification system”) present in a liquid biopsy sample is provided. This system may include an optical imaging system and a processing system. The liquid biopsy sample may include a biological structure(s). The biological structure(s) may be labeled with a fluorophore.

In another aspect, the optical imaging system may include a carrier suitable for supporting the liquid biopsy sample (“liquid biopsy sample carrier”) for the identification of the biological structure(s); an illumination system capable of illuminating the liquid biopsy sample at a specific wavelength (or wavelengths); a light detection system; and a light controlling system. The light controlling system may be configured to allow detection of emitted electromagnetic radiation from the liquid biopsy sample; allow detection of electromagnetic radiation scattered by, reflected by, and/or transmitted through the liquid biopsy sample; and guide electromagnetic radiation from the illumination system to the liquid biopsy sample, and from the liquid biopsy sample to the light detection system.

In another aspect, the biological structure identification system may be configured to receive a liquid biopsy sample by using the liquid biopsy sample carrier; illuminate the liquid biopsy sample with an electromagnetic radiation that has a specific wavelength and can be absorbed by the fluorophore; detect and determine an intensity and a wavelength of fluorescence emitted by the fluorophore; generate an image of the biological structure(s); detect and determine a morphology of each biological structure using each generated image; identify type of each biological structure based on a specific morphology and a specific fluorescence wavelength of each biological structure; form a biological structure identification bucket (“identification bucket”) based on the identified biological structure type, wherein each biological structure identification bucket contains the biological structure(s) that are similar in type; and form a set of identification buckets (“identification bucket set”) based on the identification buckets.

In another aspect, the biological structure may be a structure with a membrane, a protein, DNA, RNA, or a combination thereof. The structure with a membrane may be a cell, a vesicle, or a combination thereof. The vesicle may be an oncosome. The oncosome may have a characteristic size (e.g. characteristic length or characteristic diameter) equal to or larger than one micrometer. The oncosome may have a characteristic size (e.g. characteristic length or characteristic diameter) larger than an exosome.

In another aspect, the liquid biopsy sample may be a non-solid biological sample. The liquid biopsy sample may be a body fluid sample. The liquid biopsy sample may include a blood sample, a bone marrow sample, a peritoneal fluid sample, a urine sample, a saliva, a vaginal fluid sample, a semen sample, a tear sample, a mucus sample, an aqueous humor sample, cerebrospinal fluid (CSF) sample, or a combination thereof. The liquid biopsy sample may include a blood sample. The liquid biopsy sample may include common immune cells and rare biological structures.

In another aspect, the rare biological structures may include cancer cells that have cancer genomic profiles and/or cancer protein markers; tumor microenvironment cells that leak into circulation, wherein these cells comprise epithelial cells, endothelial cells, mesenchymal cells, other stromal cells, cells that are in various transitional states, or a mixture thereof; immune cells that are responding to the tumor itself or cancer treatment; vesicles, or a mixture thereof. The rare biological structures may include conventional circulating tumor cells, which are CK+, vimentin−, CD31− and CD45−; circulating tumor cells, which are CK+, CD31−, CD45−, and vimentin+, and wherein tumor cells may putatively in epithelial to mesenchymal transition; tumor cells, which are CK+, and coated with platelets, which are CD31+; endothelial cells, which are CD31+, vimentin+, and CK−; endothelial cells, which are CD31+, vimentin+ and CK+; megakaryocytes, which are CD31+ and vimentin−, wherein megakaryocytes may include large cells containing a single, large, multi-lobulated, polyploidy nucleus responsible for the production of blood thrombocytes platelets; large cells, which are CD31+, and CK+, wherein these large cells may be present in the liquid biopsy samples obtained from a bone marrow; cells, which are DAPI+ and vimentin+; round cells, which are CD45+, and CK+; round cells, which are CD45+, vimentin+, and CK+; clusters of cells (“cell cluster”) comprising at least two cells, wherein the cells are same type of cells and/or different types of cells; cells, which are DAPI+, CD45−, CD31−, and CK−; immune cells, which are CD45+ and vimentin−; immune cells, which are CD45+ and vimentin+(type III intermediate filament protein), extra-cellular vesicles, or a mixture thereof.

In another aspect, the liquid biopsy sample may include common biological structures and rare biological structures, wherein a total number of biological structures is a sum of number of the common biological structures and the number of rare biological structures, and wherein fraction of the rare biological structures are equal to or less than 10%, 5%, 1%, 0.1%, or 0.01% of the total number of biological structures.

In another aspect, the optical imaging system may include a fluorescence imaging system, a brightfield imaging system, or a combination thereof. The optical imaging system may include a fluorescence microscope, a brightfield microscope, or a combination thereof.

In another aspect, the emitted electromagnetic radiation may be a fluorescent radiation.

In another aspect, the processing system may include a control system, a hardware processor, a memory system, and an information conveying system.

In another aspect, the biological structure identification system may have at least one fluorescence channel. In a refinement, the biological structure identification system includes from 1 to 10 fluorescence channels. In a further refinement, the biological structure identification system includes from 4 to 7 fluorescence channels.

In another aspect, the number of fluorescence channels may be only four. These four fluorescence channels may be a first fluorescence channel configured for detection useful for nuclear segmentation and characterization; a second fluorescence channel configured to detect a cytokeratin (CK) for its epithelial-like phenotype; a third fluorescence channel configured to detect a vimentin for its endothelial/mesenchymal-like phenotype; and a fourth fluorescence channel configured to detect both a CD31 for its endothelial-like phenotype, and a CD45 for its immune cell phenotype. These four fluorescence channels may be a first fluorescence channel configured for detection of fluorescence emission at a blue color wavelength region; a second fluorescence channel configured for detection of fluorescence emission at a red color wavelength region; a third fluorescence channel configured for detection of fluorescence emission at a orange color wavelength region; and a fourth fluorescence channel configured for detection of fluorescence emission at a green color wavelength region. The first immunofluorescence channel may be configured to detect 4′,6-diamidino-2-phenylindole (DAPI) for nuclear segmentation and characterization.

In another aspect, the systems of this disclosure may be configured to identify endothelial cells and immune cells from a plurality of features. Since morphology of the endothelial cells and the immune cells can be determined from the plurality of features, the endothelial cells and immune cells can also be used identify the endothelial cells and immune cells. The systems of this disclosure may be configured to identify the endothelial cells and the immune cells from the plurality of features (and/or from the morphology of the endothelial cells and the immune cells determined from the features), and to differentiate the endothelial cells from the immune cells. The endothelial cells may have more elongated morphologies as compared to the immune cells, and the immune cells may have more round morphologies as compared to the endothelial cells.

In another aspect, the morphology of the biological structure may be determined by using at least one feature of the biological structure's morphology. The morphology of the biological structure may be determined by using at least 10 features, at least 100 features, at least 500 features, or at least 1,000 features of the biological structure's morphology. The feature may be related to size, shape, texture and structure of the biological structure's morphology.

In another aspect, the liquid biopsy sample may be obtained from a diseased human. The liquid biopsy sample may be obtained from a human afflicted with a cancer.

In another aspect, the biological structure identification system may further be configured to form a disease map based on information related to the identification bucket set(s), relate this disease map to a specific disease and disease stage, and label this disease map according to the related specific disease and its stage. The biological structure identification system may further be configured to store a disease map based on information related to the identification bucket set(s) and labeled by a disease type and the disease stage, and wherein the disease may cause formation of the biological structures forming said identification bucket set(s). The biological structure identification system is configured to form disease maps of at least two different types of diseases and stages of each disease. The biological structure identification system may further be configured to form a disease atlas of disease maps based on the disease maps of different disease types and their stages.

In another aspect, the biological structure identification system may further be configured to diagnose the disease type and its stage based on the received liquid biopsy sample from a human afflicted with a disease. The biological structure identification system may further be configured to diagnose the disease type and its stage based on a liquid biopsy sample received from a human afflicted with a disease by comparing the disease map formed for the received liquid biopsy sample with the disease maps of the disease atlas stored in the biological structure identification system prior to receiving the liquid biopsy sample.

In another aspect, the biological structure identification system may further include an information conveying system. The biological structure identification system may further be configured via the information conveying system to convey to a user an information comprising an information related to types of the biological structures present in the liquid biopsy sample, the biological structure identification buckets, the disease maps, the disease atlases, or a combination thereof.

In still another aspect, an immunofluorescence assay for analyzing a liquid biopsy sample is provided. This assay may include antibodies against cytokeratin (CK), vimentin, CD31 and CD45.

In still another aspect, a method of analyzing a liquid biopsy sample is provided. This method may include having a liquid biopsy sample comprising biological structures; preparing a sample comprising a single layer of biological structures (“single layer biological structure sample”) by using the liquid biopsy sample; using the assay(s) of this disclosure; staining the biological structures of the single layer biological structure sample by using four fluorescent components (e.g., dyes or components such as antibodies labeled with fluorophores); using the biological structure identification system(s) of this disclosure; identifying the rare biological structures through their fluorescence and morphology; and forming a biological structure identification bucket based on the identified biological structure type, wherein each biological structure identification bucket may contain a similar type of biological structures.

In yet another aspect, a method of diagnosing a disease afflicting a patient is provided. This method may include having a liquid biopsy sample from the patient comprising biological structures; preparing a sample comprising a single layer of biological structures (“single biological structure layer sample”) by using the liquid biopsy sample; staining the biological structures of the single biological structure layer sample with an assay have four fluorescent components (e.g., dyes or components such as antibodies labeled with fluorophores); using the biological structure identification system(s) of this disclosure; identifying the rare biological structures through their fluorescence and morphology; forming a biological structure identification bucket (“identification bucket”) based on the identified biological structure type, wherein each biological structure identification bucket contains the biological structure(s) that are similar in type; forming a set of identification buckets (“identification bucket set”) based on the identification buckets; comparing information related to the identification bucket set to that of the atlas; determining the disease afflicting the patient; and treating the patient.

Exemplary features of the system and the method are further disclosed through the following specific aspects:

In a first specific aspect, a system for identification of a biological structure present in a liquid biopsy sample comprises:

In a second specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the biological structure is a structure with a membrane, a protein, DNA, RNA, or a combination thereof.

In a third specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the structure with a membrane is a cell, a vesicle, or a combination thereof.

In a fourth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the vesicle is an oncosome.

In a fifth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the vesicle is an oncosome that has a characteristic size (e.g. characteristic length or characteristic diameter) equal to or larger than one micrometer.

In a sixth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the vesicle is an oncosome that has a characteristic size (e.g. characteristic length or characteristic diameter) larger than exosome.

In a seventh specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the liquid biopsy sample is a non-solid biological sample.

In an eighth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the liquid biopsy sample is a body fluid sample.

In a ninth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the liquid biopsy sample comprises a blood sample, a bone marrow sample, a peritoneal fluid sample, a urine sample, a saliva, a vaginal fluid sample, a semen sample, a tear sample, a mucus sample, an aqueous humor sample, cerebrospinal fluid (CSF) sample, or a combination thereof.

In a tenth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the liquid biopsy sample comprises a blood sample.

In an eleventh specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the liquid biopsy sample comprises common immune cells and rare biological structures.

In a twelfth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the rare biological structures are:

In a thirteenth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the rare biological structures comprise:

In a fourteenth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the liquid biopsy sample comprises common biological structures and rare biological structures, wherein a total number of biological structures is a sum of number of the common biological structures and the number of rare biological structures, and wherein fraction of the rare biological structures are equal to or less than 10%, 5%, 1%, 0.1%, or 0.01% of the total number of biological structures.

In a fifteenth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the optical imaging system comprises a fluorescence imaging system, a brightfield imaging system, or a combination thereof.

In a sixteenth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the emitted electromagnetic radiation is a fluorescent radiation.

In a seventeenth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the optical system comprises an excitation filter, an emission filter, a (dichroic) mirror, a lens, an optical fiber, or a combination thereof.

In an eighteenth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the optical imaging system comprises a fluorescence microscope, a brightfield microscope, or a combination thereof.

In a nineteenth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the processing system comprises a control system, a hardware processor, a memory system, and an information conveying system.

In a twentieth specific aspect, the system, the method, and/or the assay of any preceding or following specific aspect is such that the biological structure identification system has at least one fluorescence channel.